Methods for analyzing DNA sequence variation can be divided into two general categories: 1) genotyping for known sequence variants and 2) scanning for unknown variants. There are many methods for genotyping known sequence variants, and single step, homogeneous, closed tube methods that use fluorescent probes are available (Lay M J, et al., Clin. Chem 1997; 43:2262-7). In contrast, most scanning techniques for unknown variants require gel electrophoresis or column separation after PCR. These include singlestrand conformation polymorphism (Orita O, et al., Proc Natl Acad Sci USA 1989; 86:2766-70), heteroduplex migration (Nataraj A J, et al., Electrophoresis 1999; 20:1177-85), denaturing gradient gel electrophoresis (Abrams E S, et al., Genomics 1990; 7:463-75), temperature gradient gel electrophoresis (Wartell R M, et al., J Chromatogr A 1998; 806:169-85), enzyme or chemical cleavage methods (Taylor G R, et al., Genet Anal 1999; 14:181-6), as well as DNA sequencing. Identifying new mutations by sequencing also requires multiple steps after PCR, namely cycle sequencing and gel electrophoresis. Denaturing high-performance liquid chromatography (Xiao W, et al., Hum Mutat 2001; 17:439-74) involves injecting the PCR product into a column.
Single nucleotide polymorphisms (SNPs) are by far the most common genetic variations observed in man and other species. In these polymorphisms, only a single base varies between individuals. The alteration may cause an amino acid change in a protein, alter rates of transcription, affect mRNA spicing, or have no apparent effect on cellular processes. Sometimes when the change is silent (e.g., when the amino acid it codes for does not change), SNP genotyping may still be valuable if the alteration is linked to (associated with) a unique phenotype caused by another genetic alteration.
There are many methods for genotyping SNPs. Most use PCR or other amplification techniques to amplify the template of interest. Contemporaneous or subsequent analytical techniques may be employed, including gel electrophoresis, mass spectrometry, and fluorescence. Fluorescence techniques that are homogeneous and do not require the addition of reagents after commencement of amplification or physical sampling of the reactions for analysis are attractive. Exemplary homogeneous techniques use oligonucleotide primers to locate the region of interest and fluorescent labels or dyes for signal generation. Various PCR-based methods are completely closed-tubed, using a thermostable enzyme that is stable to DNA denaturation temperature, so that after heating begins, no additions are necessary.
Several closed-tube, homogeneous, fluorescent PCR methods are available to genotype SNPs. These include systems that use FRET oligonucleotide probes with two interacting chromophores (adjacent hybridization probes, TaqMan® probes, Molecular Beacons, Scorpions), single oligonucleotide probes with only one fluorophore (Gquenching probes, Crockett, A. O. and C. T. Wittwer, Anal. Biochem. 2001; 290:89-97 and SimpleProbes®, Idaho Technology), and techniques that use a dsDNA dye instead of covalent, fluorescently-labeled oligonucleotide probes.
PCR methods that monitor DNA melting with dsDNA fluorescent dyes have become popular in conjunction with real-time PCR. Because PCR produces enough DNA for fluorescent melting analysis, both amplification and analysis can be performed in the same tube, providing a homogeneous, closed-tube system that requires no processing or separation steps. dsDNA dyes are commonly used to identify products by their melting temperature, or Tm.
The power of DNA melting analysis depends on its resolution. Studies with UV absorbance often required hours to collect high-resolution data at rates of 0.1-1.0° C./min to ensure equilibrium. In contrast, fluorescent melting analysis is often acquired at 0.1-1.0° C./sec and resolution is limited to 2-4 points/° C. With recent advances in electronics (e.g., 24-bit A-to-D converters), high-resolution melting can be performed rapidly with 10-100 times the data density (50-100 points/° C.) of conventional real-time PCR instruments, as recently demonstrated for probe and PCR product melting. Furthermore, saturating DNA dyes, such as LCGreen® Plus (Idaho Technology, Salt Lake City, Utah), that maximize detection of mismatched duplexes (heteroduplexes) are now available (see, e.g. U.S. Patent Publication Nos. 2005/0233335 and 2006/0019253, herein incorporated by reference in their entireties). These two developments dramatically increase the power of fluorescence-based DNA melting for robust identification of single-base changes within PCR products.
High-resolution melting analysis for gene scanning relies primarily on the shape of the melting transition of the PCR products. An available method for screening for heterozygous single nucleotide polymorphisms (SNPs) within products up to 1,000 bp has a sensitivity and specificity of 97% and 99%, respectively. In many cases, high-resolution analysis of the melting transition also allows genotyping without probes. Even greater specificity for variant discrimination over a smaller region can be obtained by using unlabeled probes. Specific genotypes are inferred by correlating sequence alterations under the probe to changes in the probe Tm. With the recent advances with dyes and instrumentation, high-resolution gene scanning and genotyping with unlabeled probes can optionally be done simultaneously in the same reaction. Both PCR product and probe melting transitions may be observed in the presence of a saturating DNA dye. In addition to screening for any sequence variant between the primers in the PCR product, common polymorphisms and mutations can be genotyped. Furthermore, unbiased, hierarchal clustering can accurately group the melting curves into genotypes. One, two, or even more unlabeled probes can be used in a single PCR.
In simultaneous genotyping and scanning, product melting analysis detects sequence variants anywhere between two primers, while probe melting analysis identifies variants under a probe. If a sequence variant is between the primers and under a probe, both the presence of a variant and its genotype are obtained. If product melting indicates a variant but the probe does not, then the variation likely occurs between the primers but not under the probe, and further analysis for genotyping is necessary. Probes can be placed at sites of common sequence variation so that in most cases, if product scanning is positive, the probes will identify the sequence variants, greatly reducing the need for sequencing. With one probe, the genotype of an SNP can be established by both PCR product and probe melting. With two probes, two separate regions of the sequence can be interrogated for genotype and the rest of the PCR product scanned for rare sequence variants. Multiple probes can be used if they differ in melting temperature and if each allele presents a unique pattern of probe and/or product melting.
In one illustrative example, a population is screened for cystic fibrosis mutations. Since only 3.8% of Caucasians are cystic fibrosis carriers, one would expect 96.2% of randomly screened individuals to be negative by complete (exon and splice site) sequencing. With 27 exons, the percentage of sequencing runs expected to be positive is less than 0.14%. That is, only about 1 in a 1000 sequencing runs would be useful. This is why sequencing is not recommended for cystic fibrosis screening. Instead, a selected mutation panel is usually performed that detects 83.7% of cystic fibrosis alleles.
Alternatively, consider simultaneous scanning and genotyping for cystic fibrosis screening by high-resolution melting. If the amplicon length is kept under 400 bp, the sensitivity of high-resolution scanning approaches 100.0%. If common mutations and polymorphisms are analyzed with unlabeled probes in the same reaction, then about 80% of mutations will also be genotyped. Compared to screening by de novo sequencing, the sequencing burden can be reduced by 99.97%.
Closed-tube genotyping methods that use melting analysis have the capacity to scan for unexpected variants. Melting methods also use less complex and fewer probes than allele specific methods that require one probe for each allele analyzed. Allele discrimination by Tm or curve shape is an interesting option to fluorescent color. Dyes that generically stain double-stranded DNA are attractive for simplicity and cost. Although the reliability of genotyping by amplicon melting is controversial, a recent study found that 21 out of 21 heteroduplex pairs tested were distinguishable by high-resolution melting of small amplicons (Graham R, Liew M, Meadows C, Lyon E, Wittwer C T. Distinguishing different DNA heterozygotes by high-resolution melting. Clin Chem 2005; 51).
Although common sequence variants can usually be genotyped with one or two unlabeled probes in the same reaction, more than two probes and/or sequential reactions can also be used. For example, multiple overlapped probes can locate unexpected rare variants to within the region covered by one probe. Additional probes can be designed to identify the exact position and sequence of the variation. However, DNA sequencing is a more direct approach for identifying new, previously unknown variations, particularly when the amplified region is highly variable. Nevertheless, in the vast majority of genetic analysis, the amplified wild type sequence is known and potential common variants are limited. In these cases, scanning and genotyping can be performed in one step by DNA melting with simple oligonucleotides. No fluorescent probes or separations are required, and both amplification (15 min) and melting analysis (1-2 min) can be rapid.
As discussed above, simultaneous genotyping and scanning, as well as other genotyping techniques that employ melting analysis have been promising areas of research. However, the melting curve analysis prior to high-resolution capabilities provided a lack of specificity and accuracy. With the advent of high-resolution melting curve analysis, background fluorescence noise can interfere with the use of melting curves to accurately genotype SNPs, detect sequence variations, and detect mutations. Depending on the amplicon, previous background fluorescence removal techniques have led to some erroneous calls. By example, the baseline technique uses linear extrapolation as a method for normalizing melting curves and removing background fluorescence. This technique works well with labeled probes. However, this and other previous normalization techniques have not worked as well with unlabeled probes (Zhou L, Myers A N, Vandersteen J G, Wang L, Wittwer C T. Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye. Clin Chem. 2004; 50:1328-35) multiplex small amplicon melting (Liew M, Nelson L, Margraf R, Mitchell S, Erali M, Mao R, Lyon E, Wittwer C T. Genotyping of human platelet antigens 1-6 and 15 by high-resolution amplicon melting and conventional hybridization probes. J Mol Diag, 2006; 8:97-104) and combined amplicon and unlabeled probe melting (Zhou L, Wang L, Palais R, Pryor R, Wittwer C T). High-resolution melting analysis for simultaneous mutation scanning and genotyping in solution. Clin Chem 2005; 51:1770-7, hereby incorporated by reference), nor do they work as well for small amplicons. At least in part, this is because unlabeled probe and small amplicon melting methods often require background subtraction at lower temperatures (40-80° C.) then is usual for standard amplicon melting at 80-95° C. At these lower temperatures, the low temperature baseline is not linear, but a curve with rapidly increasing fluorescence at low temperatures. When linear extrapolation is used, the lines can intersect before the melting transition is complete, and when this occurs the previous techniques do not provide the most accurate means for melting curve analysis, in part due to their mathematical reliance on absolute fluorescence.
It would be advantageous for a system and method to genotype SNPs, detect sequence variations, and/or detect mutations with high accuracy in double stranded nucleic acids through use of high resolution melting profile techniques. It would be further advantageous if the background fluorescence could be automatically and accurately separated from a double stranded nucleic acid sample melting profile. It would be a further advantage if the system and method performed accurate melting curve analysis for small and large amplicons, as well as with unlabeled probes.